Project Summary/Abstract Platelets are anucleate bodies that circulate in the bloodstream and play a very important role in vascular hemostasis. Platelets circulate in a quiescent state in intact blood vessels but they adhere to and become activated by exposed extracellular matrix in a damaged vessel. Activated platelets spread out and bind to one another (i.e., form a thrombus), so as to close up the damaged area and initiate wound healing. Excessive bleeding occurs when platelets are deficient or hypo-responsive and pathological thrombus formation, which can result in occlusion of blood vessels and cause myocardial infarction or stroke, occurs when platelets are hyper-reactive. Because the extent of platelet activation is such an important determinant of vascular pathology, it is very important to understand how platelet activation and aggregation are regulated. The platelet contains several cell surface and intracellular proteins that coordinate transmission of activating and inhibitory signals into the platelet interior, and it is the balance of stimulatory and inhibitory cues that ultimately determines the platelet activation state. Whereas much has been learned in recent years regarding the platelet receptors and signaling cascades that contribute to platelet activation, key components of which are members of the Src Family of protein tyrosine Kinases (SFK), the molecules and pathways responsible for keeping platelet activation held in check remain poorly defined. We and others have previously demonstrated that Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, also called CD31) and the SFK, Lyn, are negative regulators of platelet activation. Previous studies in our laboratory have also begun to characterize, in platelets, a pathway by which C-terminal Src kinase (Csk) is recruited to sites of SFK activity by Csk Binding Proteins (CBP), so that Csk may carry out its important role as a negative regulator of SFK activity. In particular, our preliminary studies have revealed that a member of the Downstream of kinase (Dok) family, Dok-2, is a CBP in platelets. The overall goal of this new grant application is to develop a more complete list of inhibitory molecules in platelets, to thoroughly characterize the signaling pathways in which these molecules function, and to improve our understanding of how these molecules and pathways interact with one another to ultimately influence the platelet activation state. Specifically, over the next three-year period, we propose to: (1) determine the contribution of the inhibitory SFK, Lyn, to the inhibitory function of PECAM-1 and (2) determine how Csk binding to Dok-2 contributes to negative regulation of platelet activation. Together, these studies comprise a coordinated, focused research program designed to improve our understanding of negative regulation of platelet activation by identifying, characterizing, and examining the interactions between inhibitory receptors and signaling molecules in platelets, such as PECAM-1, Lyn, and Dok-2. We expect that information derived from this investigation has the potential to lead to improved diagnosis and treatment of bleeding disorders, myocardial infarction and stroke. Project Narrative Platelets circulate in the bloodstream and become activated when they encounter a damaged blood vessel. Activated platelets spread out and bind to one another so as to close up the damaged area and initiate wound healing. Our studies are designed to determine how platelet activation is controlled because excessive bleeding occurs when platelets are not active enough and heart attacks or strokes can occur when platelets are too active.